Gene therapy for HIV-1 infection is an option and perhaps an adjuvant to conventional drug therapy. We have developed an RNA based therapeutic strategy that utilizes hammerhead ribozymes targeted to the tat and rev genes of HIV-1. The genes encoding the ribozymes have been expressed as part of a polycistronic transcript originating form the LTR of an LN based retroviral vector. In pre-clinical trials primary hematopoietic progenitor cells transduced with the ribozyme based vectors gave rise to monocytic cells which are resistant to HIV-1 challenge. This vector- ribozyme combination is currently being utilized in a phase I clinical trial of HIV-1 infected individuals infected with autologous ribozyme and vector transduced CD34 cells. The results obtained thus far have demonstrated limited marking with the ribozyme and vector constructs. Those early trials did not monitor efficacy. In the proposed clinical trials, efficacy, and hence long term expression of the ribozymes will be an important issue. In this project we will be developing improved systems for expressing ribozyme and Rev binding aptamers from the backbone of a modified amphotropic retroviral vector, MND neo. Several different Pol II and Pol III promoters, and combinations of ribozymes and Rev binding aptamers will be examined for levels and duration of expression and anti- viral activity. New ribozyme targets will be identified using a facile method for ribozyme site selection on native mRNAs in cell extracts. These new ribozymes trials: Combinations of promoter, ribozyme, and aptamers will be tested in long term bone marrow cultures and a SCID-hu mouse. The most promising cassettes will be channeled into project 3. Constructs in the MND vector which show efficacy and long term expression will be tested in project 1 and ultimately utilized in the human clinical trial of project 4. The long range objectives of this project are the development of expression systems for therapeutic RNAs which are applicable to treatment of HIV-1 in a gene therapy setting.